ABSTRACT The mammalian adaptive immune system protects its host against infectious diseases as well as tumors in a highly specific manner. At the core of ab T lymphocyte recognition is self- vs. non-self-discrimination, a functionality endowed by clonal cell-surface T cell receptors (TCRs). The millions of distinct TCRs expressed in the mammalian thymus create a repertoire that is refined to eliminate unwanted autoreactive specificities prior to export into the peripheral lymphoid compartment. Once there, mature abT cells scan their environment during immune surveillance, generating tensile and shear stresses over a wide range of pN-nN forces. Direct evidence that the TCR acts as a mechanosensor has been provided, explaining its exquisite specificity and sensitivity yet low affinity for ligand in the absence of physical load. Recently, we showed that force-based abTCR discrimination extended to its developmental precursor, the preTCR, a pTa-b heterodimer. Moreover, reversible structural rearrangements necessary for strengthened ligand binding under force were observed in both TCR and preTCR. In this proposal, we shall combine single molecule (SM) and single cell (SMSC) methods using optical traps, structure-function mutational analyses, recombinant protein expression and molecular dynamic simulation to probe TCR and preTCR complexes with pMHC under load to provide a clear understanding for the structural basis of mechanosensing. It is our hypothesis that binding is gated for unloaded TCRs but that TCRs enter a binding reading state when force loaded, extend and either stabilize the bond with lifetime lengthening to facilitate signaling or, alternatively, quickly release from irrelevant ligands. In Aim 1, we will elucidate the critical TCR a and b subunit variable (V) and constant (C) domain structural elements including the Cb FG loop involved in mechanically modulating the strength of loaded TCR-pMHC interactions. Topologically stabilized structures as well as de-stabilizing mutations will be assessed for their ability to alter pMHC bond lifetime and conformational change as well as to impact ab T cell activation as measured by cytokine production. We will leverage newly developed single molecule and single tether assays for direct comparison of strength of loaded TCRs on isolated TCRab-pMHC complexes and ab T cell lines. Aim 2 will examine mechanical tuning of the preTCR and how preTCR pTa-b structures differ from those of TCRab. In both Aims 1 and 2, we will identify conformational transitions leading to bond strengthening and release pathways critical to T cell activation and development. The effects of preTCR mutations on thymocyte developmental progression will be determined experimentally using the thymic stromal cell line OP9-DL4 and fetal liver hematopoietic progenitors transduced with wild-type or mutant preTCRs. Aim 3 will employ in silico molecular dynamics simulation to identify unfolding pathways of TCR-pMHC or preTCR-pMHC complexes and their impact on the dynamic adaptability of the interface under load. We will thereby reveal atomistic mechanisms for mechanosensing.